Inhibitors of p38

ABSTRACT

The present invention relates to inhibitors of p38, a mammalian protein kinase involved cell proliferation, cell death and response to extracellular stimuli. The invention also relates to methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors of the invention and methods of utilizing those compositions in the treatment and prevention of various disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/298,324, filed Dec. 8, 2005, which is a continuation of U.S. patentapplication Ser. No. 10/622,320, filed Jul. 17, 2003, now U.S. Pat. No.7,205,305, which is a continuation of U.S. patent application Ser. No.09/336,266, filed Jun. 18, 1999, now U.S. Pat. No. 6,608,060, which is acontinuation of International Application No. PCT/US97/23392, filed Dec.17, 1997, which is a continuation-in-part of U.S. patent applicationSer. No. 08/862,925, filed Jun. 10, 1997, now U.S. Pat. No. 6,147,080,which is a continuation-in-part of U.S. patent application Ser. No.08/822,373, filed Mar. 20, 1997, now U.S. Pat. No. 5,945,418, whichclaims the benefit of U.S. Provisional Application No. 60/034,288, filedDec. 18, 1996, now abandoned, the disclosures of which are eachincorporated herein by reference in their entireties.

TECHNICAL FIELD OF INVENTION

The present invention relates to inhibitors of p38, a mammalian proteinkinase involved cell proliferation, cell death and response toextracellular stimuli. The invention also relates to methods forproducing these inhibitors. The invention also provides pharmaceuticalcompositions comprising the inhibitors of the invention and methods ofutilizing those compositions in the treatment and prevention of variousdisorders.

BACKGROUND OF THE INVENTION

Protein kinases are involved in various cellular responses toextracellular signals. Recently, a family of mitogen-activated proteinkinases (MAPK) have been discovered. Members of this family are Ser/Thrkinases that activate their substrates by phosphorylation [B. Stein etal., Ann. Rep. Med. Chem., 31, pp. 289-98 (1996)]. MAPKs are themselvesactivated by a variety of signals including growth factors, cytokines,UV radiation, and stress-inducing agents.

One particularly interesting MAPK is p38. p38, also known as cytokinesuppressive anti-inflammatory drug binding protein (CSBP) and RK, wasisolated from murine pre-B cells that were transfected with thelipopolysaccharide (LPS) receptor CD14 and induced with LPS. p38 hassince been isolated and sequenced, as has the cDNA encoding it in humansand mouse. Activation of p38 has been observed in cells stimulated bystresses, such as treatment of lipopolysaccharides (LPS), UV,anisomycin, or osmotic shock, and by cytokines, such as IL-1 and TNF.

Inhibition of p38 kinase leads to a blockade on the production of bothIL-1 and TNF. IL-1 and TNF stimulate the production of otherproinflammatory cytokines such as IL-6 and IL-8 and have been implicatedin acute and chronic inflammatory diseases and in post-menopausalosteoporosis [R. B. Kimble et al., Endocrinol., 136, pp. 3054-61(1995)].

Based upon this finding it is believed that p38, along with other MAPKs,have a role in mediating cellular response to inflammatory stimuli, suchas leukocyte accumulation, macrophage/monocyte activation, tissueresorption, fever, acute phase responses and neutrophilia. In addition,MAPKs, such as p38, have been implicated in cancer, thrombin-inducedplatelet aggregation, immunodeficiency disorders, autoimmune diseases,cell death, allergies, osteoporosis and neurodegenerative disorders.Inhibitors of p38 have also been implicated in the area of painmanagement through inhibition of prostaglandin endoperoxide synthase-2induction. Other diseases associated with IL-1, IL-6, IL-8 or TNFoverproduction are set forth in WO 96/21654.

Others have already begun trying to develop drugs that specificallyinhibit MAPKs. For example, PCT publication WO 95/31451 describespyrazole compounds that inhibit MAPKs, and in particular p38. However,the efficacy of these inhibitors in vivo is still being investigated.

Accordingly, there is still a great need to develop other potent,p38-specific inhibitors that are useful in treating various conditionsassociated with p38 activation.

SUMMARY OF THE INVENTION

The present invention solves this problem by providing compounds whichdemonstrate strong and specific inhibition of p38.

These compounds have the general formula:

wherein each of Q₁, and Q₂ are independently selected from 5-6 memberedaromatic carbocyclic or heterocyclic ring systems, or 8-10 memberedbicyclic ring systems comprising aromatic carbocyclic rings, aromaticheterocyclic rings or a combination of an aromatic carbocyclic ring andan aromatic heterocyclic ring.

The rings that make up Q₁ are substituted with 1 to 4 substituents, eachof which is independently selected from halo; C₁-C₃ alkyl optionallysubstituted with NR′₂, OR′, CO₂R′ or CONR′₂; O—(C₁-C₃) -alkyl optionallysubstituted with NR′₂, OR′, CO₂R′ or CONR′₂; NR′₂; OCF₃; CF₃; NO₂;CO₂R′; CONHR′; SR′; S(O₂)N(R′)₂; SCF₃; CN; N(R′)C(O)R⁴; N(R′)C(O)OR⁴;N(R′)C(O)C(O)R⁴; N(R′)S(O₂)R⁴; N(R′)R⁴; N(R⁴)₂; OR⁴; OC(O)R⁴; OP(O)₃H₂;or N═CH—N(R′)₂.

The rings that make up Q₂ are optionally substituted with up to 4substituents, each of which is independently selected from halo; C₁-C₃straight or branched alkyl optionally substituted with NR′₂, OR′, CO₂R′,S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR′₂; O—(C₁-C₃)-alkyl;O—(C₁-C₃)-alkyl optionally substituted with NR′₂, OR′, CO₂R′,S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR²; NR²; OCF₃; CF₃; NO₂; CO₂R′;CONHR′; R³; OR³; NHR³; SR³; C(O)R³; C(O)N(R′)R³; C(O)OR³; SR′;S(O₂)N(R′)₂; SCF₃; N═CH—N(R′)₂; or CN.

R′ is selected from hydrogen, (C₁-C₃)-alkyl; (C₂-C₃)-alkenyl or alkynyl;phenyl or phenyl substituted with 1 to 3 substituents independentlyselected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl orethyl.

R³ is selected from 5-6 membered aromatic carbocyclic or heterocyclicring systems.

R⁴ is (C₁-C₄)-alkyl optionally substituted with N(R′)₂, OR′, CO₂R′,CON(R′)₂, or SO₂N(R²)₂; or a 5-6 membered carbocyclic or heterocyclicring system optionally substituted with N(R′)₂, OR′, CO₂R′, CON(R′)₂, orSO₂N(R²)₂.

X is selected from —S—, —O—, —S(O₂)—, —S(O)—, —S(O₂)—N(R²)—,—N(R²)—S(O₂)—, —N(R²)—C(O)O—, —O—C(O)—N(R²), —C(O)—, —C(O)O—, —O—C(O)—,—C(O)—N(R²)—, —N(R²)—C(O)—, —N(R²)—, —C(R²)₂—, or —C(OR²)₂—.

Each R is independently selected from hydrogen, —R², —N(R²)₂, —OR², SR²,—C(O)—N(R²)₂, —S(O₂)—N(R²)₂, or —C(O)—OR², wherein two adjacent R areoptionally bound to one another and, together with each Y to which theyare respectively bound, form a 4-8 membered carbocyclic or heterocyclicring;

R² is selected from hydrogen, (C₁-C₃)-alkyl, or (C₂-C₃)-alkenyl; eachoptionally substituted with —N(R′)₂, —OR′, SR′, —C(O)—N(R′)₂,—S(O₂)—N(R′)₂, —C(O)—OR′, or R³.

Y is N or C;

A, if present, is N or CR′;

n is 0 or 1;

R₁ is selected from hydrogen, (C₁-C₃)-alkyl, OH, or O—(C₁-C₃)-alkyl.

In another embodiment, the invention provides pharmaceuticalcompositions comprising the p38 inhibitors of this invention. Thesecompositions may be utilized in methods for treating or preventing avariety of disorders, such as cancer, inflammatory diseases, autoimmunediseases, destructive bone disorders, proliferative disorders,infectious diseases, viral diseases and neurodegenerative diseases.These compositions are also useful in methods for preventing cell deathand hyperplasia and therefore may be used to treat or preventreperfusion/ischemia in stroke, heart attacks, organ hypoxia. Thecompositions are also useful in methods for preventing thrombin-inducedplatelet aggregation. Each of these above-described methods is also partof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides inhibitors of p38 having the generalformula:

wherein each of Q₁, and Q₂ are independently selected from 5-6 memberedaromatic carbocyclic or heterocyclic ring systems, or 8-10 memberedbicyclic ring systems comprising aromatic carbocyclic rings, aromaticheterocyclic rings or a combination of an aromatic carbocyclic ring andan aromatic heterocyclic ring.

The rings that make up Q₁ are substituted with 1 to 4 substituents, eachof which is independently selected from halo; C₁-C₃ alkyl optionallysubstituted with NR′₂, OR′, CO₂R′ or CONR′₂; O—(C₁-C₃) -alkyl optionallysubstituted with NR′₂, OR′, CO₂R′ or CONR′₂; NR′₂; OCF₃; CF₃; NO₂;CO₂R′; CONHR′; SR′; S(O₂)N(R′)₂; SCF₃; CN; N(R′)C(O)R⁴; N(R′)C(O)OR⁴;N(R′)C(O)C(O)R⁴; N(R′)S(O₂)R⁴; N(R′)R⁴; N(R⁴)₂; OR⁴; OC(O)R⁴; OP(O)₃H₂;or N═CH—N(R′)₂.

The rings that make up Q₂ are optionally substituted with up to 4substituents, each of which is independently selected from halo; C₁-C₃straight or branched alkyl optionally substituted with NR′₂, OR′, CO₂R′,S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR′₂; O—(C₁-C₃)-alkyl;O—(C₁-C₃)-alkyl optionally substituted with NR′₂, OR′, CO₂R′,S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR′₂; NR′₂; OCF₃; CF₃; NO₂; CO₂R′;CONHR′; R³; OR³; NHR³; SR³; C(O)R³; C(O)N(R′)R³; C(O)OR³; SR′;S(O₂)N(R′)₂; SCF₃; N═CH—N(R′)₂; or CN.

R′ is selected from hydrogen, (C₁-C₃)-alkyl; (C₂-C₃)-alkenyl or alkynyl;phenyl or phenyl substituted with 1 to 3 substituents independentlyselected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl orethyl.

R³ is selected from 5-6 membered aromatic carbocyclic or heterocyclicring systems.

R⁴ is (C₁-C₄)-alkyl optionally substituted with N(R′)₂, OR′, CO₂R′,CON(R′)₂, or SO₂N(R²)₂; or a 5-6 membered carbocyclic or heterocyclicring system optionally substituted with N(R′)₂, OR′, CO₂R′, CON(R′)₂, orSO₂N(R²)₂.

X is selected from —S—, —O—, —S(O₂)—, —S(O)—, —S(O₂)—N(R²)—,—N(R²)—S(O₂)—, —N(R²)—C(O)O—, —O—C(O)—N(R²), —C(O)—, —C(O)O—, —O—C(O)—,—C(O)—N(R²)—, —N(R²)—C(O)—, —N(R²)—, —C(R²)₂—, or —C(OR²)₂—.

Each R is independently selected from hydrogen, —R², —N(R²)₂, —OR², SR²,—C(O)—N(R²)₂, —S(O₂)—N(R²)₂, or —C(O)—OR², wherein two adjacent R areoptionally bound to one another and, together with each Y to which theyare respectively bound, form a 4-8 membered carbocyclic or heterocyclicring;

When the two R components form a ring together with the Y components towhich they are respectively bound, it will obvious to those skilled inthe art that a terminal hydrogen from each unfused R component will belost. For example, if a ring structure is formed by binding those two Rcomponents together, one being —NH—CH₃ and the other being —CH₂—CH₃, oneterminal hydrogen on each R component (indicated in bold) will be lost.Therefore, the resulting portion of the ring structure will have theformula —NH—CH₂—CH₂—CH₂—.

R² is selected from hydrogen, (C₁-C₃)-alkyl, or (C₂-C₃)-alkenyl; eachoptionally substituted with —N(R′)₂, —OR′, SR′, —C(O)—N(R′)₂,—S(O₂)—N(R′)₂, —C(O)—OR′, or R³.

Y is N or C;

A, if present, is N or CR′;

n is 0 or 1;

R₁ is selected from hydrogen, (C₁-C₃)-alkyl, OH, or O—(C₁-C₃)-alkyl. Itwill be apparent to those of skill in the art that if R₁ is OH, theresulting inhibitor may tautomerize resulting in compounds of theformula:

which are also p38 inhibitors of this invention.

According to another preferred embodiment, Q₁ is selected from phenyl orpyridyl containing 1 to 3 substituents, wherein at least one of saidsubstituents is in the ortho position and said substituents areindependently selected from chloro, fluoro, bromo, —CH₃, —OCH₃, —OH,—CF₃, —OCF₃, —O(CH₂)₂CH₃, NH₂, 3,4-methylenedioxy, —N(CH₃)₂,—NH—S(O)₂-phenyl, —NH—C(O)O—CH₂-4-pyridine, —NH—C(O)CH₂-morpholine,—NH—C(O)CH₂—N(CH₃)₂, —NH—C(O)CH₂-piperazine, —NH—C(O)CH₂-pyrrolidine,—NH—C(O)C(O)-morpholine, —NH—C(O)C(O)-piperazine,—NH—C(O)C(O)-pyrrolidine, —O—C(O)CH₂—N(CH₃)₂, or —O— (CH₂)₂—N(CH₃)₂.

Even more preferred are phenyl or pyridyl containing at least 2 of theabove-indicated substituents both being in the ortho position.

Some specific examples of preferred Q₁ are:

Most preferably, Q₁ is selected from 2-fluoro-6-trifluoromethylphenyl,2,6-difluorophenyl, 2,6-dichlorophenyl, 2-chloro-4-hydroxyphenyl,2-chloro-4-aminophenyl, 2,6-dichloro-4-aminophenyl,2,6-dichloro-3-aminophenyl, 2,6-dimethyl-4-hydroxyphenyl,2-methoxy-3,5-dichloro-4-pyridyl, 2-chloro-4,5 methylenedioxy phenyl, or2-chloro-4-(N-2-morpholino-acetamido)phenyl.

According to a preferred embodiment, Q₂ is phenyl or pyridyl containing0 to 3 substituents, wherein each substituent is independently selectedfrom chloro, fluoro, bromo, methyl, ethyl, isopropyl, —OCH₃, —OH, —NH₂,—CF₃, —OCF₃, —SCH₃, —C(O)OH, —C(O)OCH₃, —CH₂NH₂, —N(CH₃)₂,—CH₂-pyrrolidine and —CH₂OH.

Some specific examples of preferred Q₂ are:

unsubstituted 2-pyridyl or unsubstituted phenyl.

Most preferred are compounds wherein Q₂ is selected from phenyl,2-isopropylphenyl, 3,4-dimethylphenyl, 2-ethylphenyl, 3-fluorophenyl,2-methylphenyl, 3-chloro-4-fluorophenyl, 3-chlorophenyl,2-carbomethoxylphenyl, 2-carboxyphenyl, 2-methyl-4-chlorophenyl,2-bromophenyl, 2-pyridyl, 2-methylenehydroxyphenyl, 4-fluorophenyl,2-methyl-4-fluorophenyl, 2-chloro-4-fluorphenyl, 2,4-difluorophenyl,2-hydroxy-4-fluorphenyl or 2-methylenehydroxy-4-fluorophenyl.

According to yet another preferred embodiment,

X is —S—, —O—, —S(O₂)—, —S(O)—, —NR—, —C(R₂)— or —C(O)—. Mostpreferably, X is S.

According to another preferred embodiment, n is 1 and A is N.

According to another preferred embodiment, each Y is C.

According an even more preferred embodiment,

each Y is C and the R attached to those Y components is selected fromhydrogen or methyl.

Some specific inhibitors of this invention are set forth in the tablebelow.

TABLE 1 Formula Ia and Ib Compounds. cpd # structure 2

3

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

According to another embodiment, the present invention provides methodsof producing inhibitors of p38 of the formula (Ia) depicted above. Thesemethods involve reacting a compound of formula II:

wherein each of the variables in the above formula are the same asdefined above for the inhibitors of this invention, with a leaving groupreagent of formula IIa:

wherein R′ is as defined above, or a leaving group reagent of formulaIIb:

wherein each of L₁, L₂, and L₃ independently represents a leaving group.

The leaving group reagent used in this reaction is added in excess,either neat or with a co-solvent, such as toluene. The reaction iscarried out at a temperature of between 25° C. and 150° C.

Leaving group reagents of formula IIa that are useful in producing thep38 inhibitors of this invention include dimethylformamidedimethylacetal, dimethylacetamide dimethylacetal, trimethylorthoformate, dimethylformamide diethylacetal and other relatedreagents. Preferably the leaving group reagent of formula IIa used toproduce the inhibitors of this invention is dimethylformamidedimethylacetal.

Leaving group reagents of formula IIb that are useful in producing thep38 inhibitors of this invention include phosgene, carbonyldiimidazole,diethyl carbonate and triphosgene.

More preferred methods of producing the compounds of this inventionutilize compounds of formula II wherein each of the variables aredefined in terms of the more preferred and most preferred choices as setforth above for the compounds of this invention.

Because the source of R₁ is the leaving group reagent (C—R′ or C═O), itsidentity is, of course, dependent on the structure of that reagent.Therefore, in compounds where R₁ is OH, the reagent used must be IIb.Similarly, when R₁ is H or (C₁-C₃)-alkyl, the reagent used must be IIa.In order to generate inhibitors wherein R′ is O—(C₁-C₃)-alkyl, acompound wherein R₁ is OH is first generated, followed by alkylation ofthat hydroxy by standard techniques, such as treatment with Na hydridein DMF, methyl iodide and ethyl iodide.

The immediate precursors to the inhibitors of this invention of formulaIa (i.e., compounds of Formula II) may themselves be synthesized byeither of the synthesis schemes depicted below:

In Scheme 1, the order of steps 1) and 2) can be reversed. Also, thestarting nitrile may be replaced by a corresponding acid or by an ester.Alternatively, other well-known latent carboxyl or carboxamide moietiesmay be used in place of the nitrile (see scheme 2). Variations such ascarboxylic acids, carboxylic esters, oxazolines or oxizolidinones may beincorporated into this scheme by utilizing subsequent deprotection andfunctionalization methods which are well known in the art

The base used in the first step of Scheme 1 (and in Scheme 2, below) isselected from sodium hydride, sodium amide, LDA, lithiumhexamethyldisilazide, sodium hexamethyldisilazide or any number of othernon-nucleophilic bases that will deprotonate the position alpha to thenitrile.

Also, the addition of HX-Q₂ in the single step depicted above may besubstituted by two steps—the addition of a protected or unprotected Xderivative followed by the addition of a Q₂ derivative in a subsequentstep.

In Scheme 2, Z is selected from COOH, COOR′, CON(R′)₂, oxazoline,oxazolidinone or CN. R′ is as defined above.

According to another embodiment, the present invention provides methodsof producing inhibitors of p38 of the formula (Ib) depicted above. Thesemethods involve reacting a compound of formula III:

wherein each of the variables in the above formula are the same asdefined above for the inhibitors of this invention, with a leaving groupreagent of formula:

as described above.

Two full synthesis schemes for the p38 inhibitors of formula (Ib) ofthis invention are depicted below.

In scheme 3, a Q₁, substituted derivative may be treated with a basesuch as sodium hydride, sodium amide, LDA, lithium hexamethyldisilazide,sodium hexamethyldisilazide or any number of other non-nucleophilicbases to deprotonate the position alpha to the Z group, which representsa masked amide moiety. Alternatively, Z is a carboxylic acid, carboxylicester, oxazoline or oxazolidinone. The anion resulting fromdeprotonation is then contacted with a nitrogen bearing heterocycliccompound which contains two leaving groups, or latent leaving groups, inthe presence of a Palladium catalyst. One example of such compound maybe 2,6-dichloropyridine.

In step two, the Q₂ ring moiety is introduced. This may be performedutilizing many reactions well known in the art which result in theproduction of biaryl compounds. One example may be the reaction of anaryl lithium compound with the pyridine intermediate produced in step 1.Alternatively, an arylmetallic compound such as an aryl stannane or anaryl boronic acid may be reacted with the aryl halide portion of thepyridine intermediate in the presence of a Pd^(o) catalyst.

In step 3 the Z group is deprotected and/or functionalized to form theamide compound. When Z is a carboxylic acid, carboxylic ester, oxazolineor oxazolidinone, variations in deprotection and functionalizationmethods which are well known in the art are employed to produce theamide. Finally in step 4, the amide compound is cyclized to the finalproduct utilizing reagents such as DMF acetal or similar reagents eitherneat or in an organic solvent.

Scheme 4 is similar except that the a biaryl intermediate is firstgenerated prior to reaction with the Q1 starting material.

According to another embodiment, the invention provides inhibitors ofp38 similar to those of formulae Ia and Ib above, but wherein the C═N inthe ring bearing the Q₁ substituent is reduced. These inhibitors havethe formula:

wherein A, Q₁, Q₂, R′, R′, X, Y and n are defined in the same manner asset forth for compounds of formulae Ia and Ib. These definitions holdfor all embodiments of each of these variables (i.e., basic, preferred,more preferred and most preferred). R⁵ is selected from hydrogen,—CR′₂OH, —C(O)R⁴, —C(O)OR⁴, —CR′₂OPO₃H₂, and salts of —PO₃H₂.

When R⁵ is not hydrogen, the resulting compounds are expected to beprodrug forms which should be cleaved in vivo to produce a compoundwherein R⁵ is hydrogen.

According to other preferred embodiments, in compounds of formula Ic, Ais preferably nitrogen, n is preferably 1, and X is preferably sulfur.In compounds of formula Ic or Id, Q₁ and Q₂ are preferably the samemoieties indicated above for those variables in compounds of formulae Iaand Ib.

Compounds of formulae Ic and Id may be prepared directly from compoundsof formulae Ia or Ib which contain a hydrogen, C₁-C₃ alkyl or C₂-C₃alkenyl or alkynyl at the R₁ position (e.g., where R₁=R′). The synthesisschemes for these compounds is depicted in Schemes 5 and 6, below.

In these schemes, compounds of formula Ia or Ib are reduced by reactionwith an excess of diisobutylaluminum hydride, or equivalent reagent toyield the ring reduced compounds of formula Ic or Id, respectively.

The addition of an R⁵ component other than hydrogen onto the ringnitrogen is achieved by reacting the formula Ic or Id compoundsindicated above with the appropriate reagent(s). Examples of suchmodifications are provided in the Example section below.

Some specific inhibitors of this invention of formula Ic are set forthin the table below.

TABLE 2 Formula Ic Compounds. cpd # structure 101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

According to yet another embodiment, the invention provides p38inhibitors of the formulae:

wherein A, Q₁, Q₂, R′, X, Y and n are defined in the same manner as setforth for compounds of formulae Ia and Ib. These definitions hold forall embodiments of each of these variables (i.e., basic, preferred, morepreferred and most preferred). More preferably, in compounds of formulaIe, Q₂ is unsubstituted phenyl.

Q₃ is a 5-6 membered aromatic carbocyclic or heterocyclic ring system,or an 8-10 membered bicyclic ring system comprising aromatic carbocyclicrings, aromatic heterocyclic rings or a combination of an aromaticcarbocyclic ring and an aromatic heterocyclic ring. The rings of Q₃ aresubstituted with 1 to 4 substituents, each of which is independentlyselected from halo; C₁-C₃ alkyl optionally substituted with NR′₂, OR′,CO₂R′ or CONR′₂; O—(C₁-C₃)-alkyl optionally substituted with NR′₂, OR′,CO₂R′ or CONR′₂; NR′₂; OCF₃; CF₃; NO₂; CO₂R′; CONHR′; SR′; S(O₂)N(R′)₂;SCF₃; CN; N(R′)C(O)R⁴; N(R′)C(O)OR⁴; N(R′)C(O)C(O)R⁴; N(R′)S(O₂)R⁴;N(R′)R⁴; N(R⁴)₂; OR⁴; OC(O)R⁴; OP(O)₃H₂; or N═CH—N(R′)₂.

According to one preferred embodiment, Q₃ is substituted with 2 to 4substituents, wherein at least one of said substituents is present inthe ortho position relative to the point of attachment of Q₃ to the restof the inhibitor. When Q₃ is a bicyclic ring, the 2 substituents in theortho position are present on the ring that is closest (i.e., directlyattached) to the rest of the inhibitor molecule. The other two optionalsubstituents may be present on either ring. More preferably, both suchortho positions are occupied by one of said substituents.

According to another preferred embodiment, Q₃ is a monocycliccarbocyclic ring, wherein each ortho substituent is independentlyselected from halo or methyl. According to another preferred embodiment,Q₃ contains 1 or 2 additional substituents independently selected fromNR′₂, OR′, CO₂R′CN, N(R′)C(O)R⁴; N(R′)C(O)OR⁴; N(R′)C(O)C(O)R⁴;N(R′)S(O₂)R⁴; N(R′)R⁴; N(R⁴)₂; OR⁴; OC(O)R⁴; OP(O)₃H₂; or N═CH—N(R′)₂.

Preferably, Q₃ is selected from any of the Q₃ moieties present in the Iecompounds set forth in Table 3, below, or from any of the Q₃ moietiespresent in the Ig compounds set forth in Table 4, below.

Those of skill will recognize compounds of formula Ie as being thedirect precursors to certain of the formula Ia and formula Ic p38inhibitors of this invention (i.e., those wherein Q₁=Q₃). Those of skillwill also recognize that compounds of formula Ig are precursors tocertain of the formula Ib and Id p38 inhibitors of this invention (i.e.,those wherein Q₁=Q₃). Accordingly, the synthesis of formula Ieinhibitors is depicted above in Schemes 1 and 2, wherein Q₁ is replacedby Q₃. Similarly, the synthesis of formula Ig inhibitors is depictedabove in Schemes 3 and 4, wherein Q₁ is replaced by Q₃.

The synthesis of formula If and formula Ih inhibitors is depicted belowin Schemes 7 and 8.

Scheme 8 depicts the synthesis of compounds of type Ih. For example,treating an initial dibromo derivative, such as 2,6 dibromopyridine,with an amine in the presence of a base such as sodium hydride yieldsthe 2-amino-6-bromo derivative. Treatment of this intermediate with aphenylboronic acid analog (a Q2-boronic acid) such as phenyl boronicacid in the presence of a palladium catalyst gives the disubstitutedderivative which can then be acylated to the final product. The order ofthe first two steps of this synthesis may be reversed.

Without being bound by theory, applicants believe that the diorthosubstitution in the Q₃ ring of formula Ie and Ig inhibitors and thepresence of a nitrogen directly attached to the Q₁ ring in formula Ifand Ih inhibitors causes a “flattening” of the compound that allows itto effectively inhibit p38.

A preferred formula Ie inhibitor of this invention is one wherein A iscarbon, n is 1, X is sulfur, each Y is carbon, each R is hydrogen, Q₃ is2,6-dichlorophenyl and Q₂ is phenyl, said compound being referred to ascompound 201. A preferred formula Ig inhibitor of this invention is onewherein Q₃ is 2,6-dichlorophenyl, Q₂ is phenyl, each Y is carbon andeach R is hydrogen. This compound is referred to herein as compound 202.Other preferred formula Ig compounds of this invention are those listedin Table 4, below.

Preferred Ih compounds of this invention are those depicted in Table 5,below. Other preferred Ih compounds are those wherein Q₁ is phenylindependently substituted at the 2 and 6 positions by chloro or fluoro;each Y is carbon; each R is hydrogen; and Q₂ is 2-methylphenyl,4-fluorophenyl, 2,4-difluorophenyl, 2-methylenehydroxy-4-fluorophenyl,or 2-methyl-4-fluorophenyl.

Some specific inhibitors of formulae Ie, Ig and Ih are depicted in thetables below.

TABLE 3 Formula Ie Inhibitors. cmpd # structure 201

203

204

205

206

207

208

209

TABLE 4 Formula Ig Inhibitors. cpd # structure  202/ 301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

1301 

TABLE 5 Compound Ih Inhibitors. cpd # structure 401

402

403

404

405

406

407

408

409

410

411

412

The activity of the p38 inhibitors of this invention may be assayed byin vitro, in vivo or in a cell line. In vitro assays include assays thatdetermine inhibition of either the kinase activity or ATPase activity ofactivated p38. Alternate in vitro assays quantitate the ability of theinhibitor to bind to p38 and may be measured either by radiolabellingthe inhibitor prior to binding, isolating the inhibitor/p38 complex anddetermining the amount of radiolabel bound, or by running a competitionexperiment where new inhibitors are incubated with p38 bound to knownradioligands.

Cell culture assays of the inhibitory effect of the compounds of thisinvention may determine the amounts of TNF, IL-1, IL-6 or IL-8 producedin whole blood or cell fractions thereof in cells treated with inhibitoras compared to cells treated with negative controls. Level of thesecytokines may be determined through the use of commercially availableELISAs.

An in vivo assay useful for determining the inhibitory activity of thep38 inhibitors of this invention is the suppression of hind paw edema inrats with Mycobacterium butyricum-induced adjuvant arthritis. This isdescribed in J. C. Boehm et al., J. Med. Chem., 39, pp. 3929-37 (1996),the disclosure of which is herein incorporated by reference. The p38inhibitors of this invention may also be assayed in animal models ofarthritis, bone resorption, endotoxin shock and immune function, asdescribed in A. M. Badger et al., J. Pharmacol. ExperimentalTherapeutics, 279, pp. 1453-61 (1996), the disclosure of which is hereinincorporated by reference.

The p38 inhibitors or pharmaceutical salts thereof may be formulatedinto pharmaceutical compositions for administration to animals orhumans. These pharmaceutical compositions, which comprise and amount ofp38 inhibitor effective to treat or prevent a p38-mediated condition anda pharmaceutically acceptable carrier, are another embodiment of thepresent invention.

The term “p38-mediated condition”, as used herein means any disease orother deleterious condition in which p38 is known to play a role. Thisincludes, conditions which are known to be caused by IL-1, TNF, IL-6 orIL-8 overproduction. Such conditions include, without limitation,inflammatory diseases, autoimmune diseases, destructive bone disorders,proliferative disorders, infectious diseases, neurodegenerativediseases, allergies, reperfusion/ischemia in stroke, heart attacks,angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiachypertrophy, thrombin-induced platelet aggregation, and conditionsassociated with prostaglandin endoperoxide synthase-2.

Inflammatory diseases which may be treated or prevented include, but arenot limited to acute pancreatitis, chronic pancreatitis, asthma,allergies, and adult respiratory distress syndrome. Autoimmune diseaseswhich may be treated or prevented include, but are not limited to,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis,diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, atopic dermatitis, chronic active hepatitis,myasthenia gravis, multiple sclerosis, inflammatory bowel disease,ulcerative colitis, Crohn's disease, psoriasis, or graft vs. hostdisease.

Destructive bone disorders which may be treated or prevented include,but are not limited to, osteoporosis, osteoarthritis and multiplemyeloma-related bone disorder.

Proliferative diseases which may be treated or prevented include, butare not limited to, acute myelogenous leukemia, chronic myelogenousleukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Angiogenic disorders which may be treated or prevented include solidtumors, ocular neovasculization, infantile haemangiomas.

Infectious diseases which may be treated or prevented include, but arenot limited to, sepsis, septic shock, and Shigellosis.

Viral diseases which may be treated or prevented include, but are notlimited to, acute hepatitis infection (including hepatitis A, hepatitisB and hepatitis C), HIV infection and CMV retinitis.

Neurodegenerative diseases which may be treated or prevented by thecompounds of this invention include, but are not limited to, Alzheimer'sdisease, Parkinson's disease, cerebral ischemias or neurodegenerativedisease caused by traumatic injury.

“p38-mediated conditions” also include ischemia/reperfusion in stroke,heart attacks, myocardial ischemia, organ hypoxia, vascular hyperplasia,cardiac hypertrophy, and thrombin-induced platelet aggregation.

In addition, p38 inhibitors in this invention are also capable ofinhibiting the expression of inducible pro-inflammatory proteins such asprostaglandin endoperoxide synthase-2 (PGHS-2), also referred to ascyclooxygenase-2 (COX-2). Therefore, other “p38-mediated conditions” areedema, analgesia, fever and pain, such as neuromuscular pain, headache,cancer pain, dental pain and arthritis pain.

The diseases that may be treated or prevented by the p38 inhibitors ofthis invention may also be conveniently grouped by the cytokine (IL-1,TNF, IL-6, IL-8) that is believed to be responsible for the disease.

Thus, an IL-1-mediated disease or condition includes rheumatoidarthritis, osteoarthritis, stroke, endotoxemia and/or toxic shocksyndrome, inflammatory reaction induced by endotoxin, inflammatory boweldisease, tuberculosis, atherosclerosis, muscle degeneration, cachexia,psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis,rubella arthritis, acute synovitis, diabetes, pancreatic β-cell diseaseand Alzheimer's disease.

TNF-mediated disease or condition includes, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusioninjury, graft vs. host reaction, allograft rejections, fever andmyalgias due to infection, cachexia secondary to infection, AIDS, ARC ormalignancy, keloid formation, scar tissue formation, Crohn's disease,ulcerative colitis or pyresis. TNF-mediated diseases also include viralinfections, such as HIV, CMV, influenza and herpes; and veterinary viralinfections, such as lentivirus infections, including, but not limited toequine infectious anemia virus, caprine arthritis virus, visna virus ormaedi virus; or retrovirus infections, including feline immunodeficiencyvirus, bovine immunodeficiency virus, or canine immunodeficiency virus.

IL-8 mediated disease or condition includes diseases characterized bymassive neutrophil infiltration, such as psoriasis, inflammatory boweldisease, asthma, cardiac and renal reperfusion injury, adult respiratorydistress syndrome, thrombosis and glomerulonephritis.

In addition, the compounds of this invention may be used topically totreat or prevent conditions caused or exacerbated by IL-1 or TNF. Suchconditions include inflamed joints, eczema, psoriasis, inflammatory skinconditions such as sunburn, inflammatory eye conditions such asconjunctivitis, pyresis, pain and other conditions associated withinflammation.

In addition to the compounds of this invention, pharmaceuticallyacceptable salts of the compounds of this invention may also be employedin compositions to treat or prevent the above-identified disorders.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts. Salts derived from appropriate bases include alkalimetal (e.g., sodium and potassium), alkaline earth metal (e.g.,magnesium), ammonium and N—(C₁₋₄ alkyl)⁴⁺ salts. This invention alsoenvisions the quaternization of any basic nitrogen-containing groups ofthe compounds disclosed herein. Water or oil-soluble or dispersibleproducts may be obtained by such quaternization.

Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of p38 inhibitor that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, the particular mode of administration. Preferably, thecompositions should be formulated so that a dosage of between 0.01-100mg/kg body weight/day of the inhibitor can be administered to a patientreceiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

According to another embodiment, the invention provides methods fortreating or preventing a p38-mediated condition comprising the step ofadministering to a patient one of the above-described pharmaceuticalcompositions. The term “patient”, as used herein, means an animal,preferably a human.

Preferably, that method is used to treat or prevent a condition selectedfrom inflammatory diseases, autoimmune diseases, destructive bonedisorders, proliferative disorders, infectious diseases, degenerativediseases, allergies, reperfusion/ischemia in stroke, heart attacks,angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiachypertrophy, and thrombin-induced platelet aggregation.

According to another embodiment, the inhibitors of this invention areused to treat or prevent an IL-1, IL-6, IL-8 or TNF-mediated disease orcondition. Such conditions are described above.

Depending upon the particular p38-mediated condition to be treated orprevented, additional drugs, which are normally administered to treat orprevent that condition may be administered together with the inhibitorsof this invention. For example, chemotherapeutic agents or otheranti-proliferative agents may be combined with the p38 inhibitors ofthis invention to treat proliferative diseases.

Those additional agents may be administered separately, as part of amultiple dosage regimen, from the p38 inhibitor-containing composition.Alternatively, those agents may be part of a single dosage form, mixedtogether with the p38 inhibitor in a single composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example 1 Synthesis of p38 Inhibitor Compound 1

Examples of the synthesis of several compounds of formula Ia are setforth in the following 4 examples.

A.

To a slurry of sodium amide, 90% (1.17 g., 30 mmol) in drytetrahydrofuran (20 ml) we added a solution of benzyl cyanide (2.92 g.,25.0 mmol) in dry tetrahydrofuran (10 ml) at room temperature. Themixture was stirred at room temperature for 30 minutes. To the reactionmixture we added a solution of 3,6-dichloropyridazine (3.70 g., 25.0mmol) in dry tetrahydrofuran (10 ml). After stirring for 30 minutes, thereaction mixture was diluted with an aqueous saturated sodiumbicarbonate solution. The reaction mixture was then extracted with ethylacetate. The layers were separated and the organic was washed withwater, brine, dried over magnesium sulfate, filtered and concentrated invacuo.

The residue was purified by chromatography on silica gel (eluant: 30%ethyl acetate in n-hexane) to give 3.71 g. (16.20 mmol ˜54%) of productas a white solid.

B.

To a slurry of sodium hydride, 95% (0.14 g., 6.0 mmol) in drytetrahydrofuran (10 ml) we added thiophenol (0.66 g, 6.0 ml.) at roomtemperature. The reaction mixture was then stirred for 10 minutes. Tothe reaction mixture we added a solution of the product from step A.,above (1.31 g., 5.72 mmol) in absolute ethanol (20 ml.). The reactionmixture was then brought to reflux and stirred there for one hour. Thecool reaction mixture was concentrated in vacuo. The residue was dilutedwith a 1N sodium hydroxide solution (10 ml), then extracted withmethylene chloride. The organic phase was washed with water, brine,dried over magnesium sulfate and concentrated in vacuo.

The residue was purified by chromatography on silica gel (eluant: 20%ethyl acetate in n-hexane) to give 0.66 g. (2.19 mmol ˜40%) of productas a white solid.

C.

A mixture of the product from step B. (0.17 g., 0.69 mmol) andconcentrated sulfuric acid (5 ml) was heated to 100° C. for one hour.The solution was cooled and adjusted to pH 8 with a saturated sodiumbicarbonate solution. The reaction mixture was extracted with methylenechloride. The organic layer was washed with water, brine, dried overmagnesium sulfate and concentrated in vacuo to give 0.22 g. (0.69 mmol˜100%) of compound pre-1 as an orange oil. ¹H NMR (500 MHz, CD3OD) d7.7(d), 7.5 (d), 7.4 (m), 7.3-7.2 (m).

D.

A solution of pre-1 from step C. (0.22 g., 0.69 mmol) andN,N-dimethylformamide dimethylacetal (0.18 g., 1.5 mmol) in toluene (5ml) was heated at 100° C. for one hour. Upon cooling, the resultingsolid was filtered and dissolved in warm ethyl acetate. The product wasprecipitated with the dropwise addition of diethyl ether. The productwas then filtered and washed with diethyl ether to give 0.038 g. ofcompound 1 as a yellow solid. ¹H NMR (500 MHz, CDCl3) d8.63 (s),7.63-7.21 (m), 6.44 (d).

Example 2 Synthesis of p38 Inhibitor Compound 2

A.

The first intermediate depicted above was prepared in a similar manneras in Example 1A, using 4-fluorophenylacetonitrile, to afford 1.4 g (5.7mmol, ˜15%) of product.

B.

The above intermediate was prepared in a similar manner as in Example1B. This afforded 0.49 g (1.5 mmol, 56%) of product.

C.

The above intermediate was prepared in a similar manner as Example 1C.This afforded 0.10 g (0.29 mmol, 45%) of compound pre-2. ¹H NMR (500MHz, CDCl3) d 7.65-7.48 (m), 7.47-7.30 (m), 7.29-7.11 (m), 7.06-6.91(m), 5.85 (s, br).

D.

Compound 2 (which is depicted in Table 1) was prepared from pre-2 in asimilar manner as in Example 1D. This afforded 0.066 g of product. ¹HNMR (500 MHz, CDCl3) δ 8.60 (s), 7.62-7.03 (m), 6.44 (d)).

Example 3 Synthesis of p38 Inhibitor Compound 6

A.

The first intermediate in the preparation of compound 6 was prepared ina manner similar to that described in Example 1A, using2,6-dichlorophenyl-acetonitrile, to afford 2.49 g (8.38, 28%) ofproduct.

B.

The next step in the synthesis of compound 6 was carried out in asimilar manner as described in Example 1B. This afforded 2.82 g (7.6mmol, 91%) of product.

C.

The final intermediate, pre-6, was prepared in a similar manner asdescribed in Example 1C. This afforded 0.89 g (2.3 mmol, 85%) ofproduct. ¹H NMR (500 MHz, CD3OD) δ 7.5-7.4 (dd), 7.4 (m), 7.3 (d), 7.2(m), 7.05 (d).

D.

The final step in the synthesis of compound 6 (which is depicted inTable 1) was carried out as described in Example 1D. This afforded 0.06g of product. ¹H NMR (500 MHz, CDCl3) δ 8.69 (s), 7.65-7.59 (d),7.58-7.36 (m), 7.32-7.22 (m), 6.79 (d), 6.53 (d).

Example 4 Preparation of p38 Inhibitor Compound 5

A.

The first intermediate in the synthesis of compound 5 was prepared in asimilar manner as described in Example 1A, using2,4-dichlorophenylacetonitrile, to afford 3.67 g (12.36 mmol, 49%) ofproduct.

B.

The second intermediate was prepared in a similar manner as described inExample 1B. This afforded 3.82 g (9.92 mmol, 92%) of product.

C.

The final intermediate, pre-5, was prepared in a similar manner asdescribed in Example 1C. This afforded 0.10 g (0.24 mmol, 92%) ofproduct. ¹H NMR (500 MHz, CD3OD) δ 7.9 (d), 7.7 (d), 7.6-7.5 (dd),7.4-7.3 (m), 2.4 (s).

D.

The final step in the preparation of compound 5 (which is depicted inTable 1) was carried out in a similar manner as described in Example 1D.This afforded 0.06 g of product. ¹H NMR (500 MHz, CDCl3) d 8.64 (s),7.51-7.42 (m), 7.32-7.21 (m), 6.85 (d), 6.51 (d), 2.42 (s).

Other compounds of formula Ia of this invention may be synthesized in asimilar manner using the appropriate starting materials.

Example 5 Preparation of A p38 Inhibitor Compound of Formula Ib

An example of the synthesis of a p38 inhibitor of this invention of theformula Ib is presented below.

A.

To a slurry of sodium amide, 90% (1.1 eq) in dry tetrahydrofuran wasadded a solution of 2,6-dichlorobenzyl cyanide (1.0 eq) in drytetrahydrofuran at room temperature. The mixture was stirred at roomtemperature for 30 minutes. To the reaction mixture was added a solutionof 2,6-dichloropyridine (1 eq) in dry tetrahydrofuran. The reaction wasmonitored by TLC and, when completed the reaction mixture was dilutedwith an aqueous saturated sodium bicarbonate solution. The reactionmixture was then extracted with ethyl acetate. The layers were separatedand the organic layer was washed with water, brine, dried over magnesiumsulfate, filtered and concentrated in vacuo. The residue was purified bychromatography on silica gel to yield pure product.

B.

To a solution of 4-fluoro-bromobenzene (1 eq) in dry tetrahydrofuran at−78° C. was added t-butyllithium (2 eq, solution in hexanes). Thereaction mixture was then stirred for 30 minutes. To the reactionmixture was added a solution of the product from Step A (1 eq) in dryTHF. The reaction mixture was then monitored and slowly brought to roomtemperature. The reaction mixture was quenched with water then extractedwith methylene chloride. The organic phase was washed with water, brine,dried over magnesium sulfate and concentrated in vacuo. The residue waspurified by chromatography on silica gel to yield the product.

C.

A mixture of the product step B and concentrated sulfuric acid washeated to 100° C. for one hour. The solution was cooled and adjusted topH 8 with a saturated sodium bicarbonate solution. The reaction mixturewas extracted with methylene chloride. The organic layer was washed withwater, brine, dried over magnesium sulfate and concentrated in vacuo togive product. The final product was purified by silica gel flashchromatography

D.

A solution of the product Step C (1 eq) and N,N-Dimethylformamidedimethylacetal (2 eq) in toluene is heated at 100° C. for one hour. Uponcooling, the resulting mixture is filtered and dissolved in warm ethylacetate. The product is precipitated with the dropwise addition ofdiethyl ether. The product is then filtered and washed with diethylether to give a p38 inhibitor of formula Ib. The final product isfurther purified by silica gel chromatography.

Other compounds of formula Ib of this invention may be synthesized in asimilar manner using the appropriate starting materials.

Example 6 Synthesis of p38 Inhibitor Compound 103

This example sets forth a typical synthesis of a compound of formula Ic.

A.

The p38 inhibitor compound 12 is prepared essentially as set forth forin Example 4, except that 4-fluorothiophenyl is utilized in step B.

B.

Compound 12 was dissolved in dry THF (5 ml) at room temperature. To thissolution we added diisobutylaluminum hydride (1M solution in toluene, 5ml, mmol) and the reaction was stirred at room temperature for 1 hour.The reaction mix was then diluted with ethyl acetate and quenched by theaddition of Rochelle salt. The layers were separated and the organiclayer was isolated, washed with water, washed with brine, dried overmagnesium sulfate and filtered to yield crude compound 103. The crudeproduct was chromatographed on silica gel eluting with 2% methanol inmethylene chloride. Pure compound 103 was thus obtained (210 mg, 50%yield): 1H NMR (500 Mhz, CDCl3) 7.51 (m, 1H), 7.38 (d, 2H), 7.20 (t,2H), 7.08 (t, 2H), 6.70 (broad s, 1H), 6.30 (dd, 2H), 5.20 (s, 2H).

Example 7 Synthesis of p38 Inhibitor Compound 201

A.

The starting nitrile shown above (5.9 g, 31.8 mmol) was dissolved in DMF(20 ml) at room temperature. Sodium hydride (763 mg, 31.8 mmol) was thenadded, resulting in a bright yellow-colored solution. After 15 minutes asolution of 2,5 dibromopyridine (5.0 gr., 21.1 mmol) in DMF (10 ml) wasadded followed by Palladium tetrakis (triphenylphosphine) (3 mmol). Thesolution was then refluxed for 3 hrs. The reaction was cooled to roomtemperature and diluted with ethyl acetate. The organic layer was thenisolated, washed with water and then with brine, dried over magnesiumsulfate, filtered and evaporated in vacuo to a crude oil. Flash columnchromatography eluting with 10% ethyl acetate in hexane afforded product(5.8 g, 84%) as an off white solid.

B.

The bromide produced in step A (194.8 mg, 0.57 mmol) was dissolved inxylene (15 ml). To this solution we added thiophenylstannane (200 μl,587 mmol) and palladium tetrakis (triphenylphosphine) (25 mg). Thesolution was refluxed overnight, cooled, filtered and evaporated invacuo. The crude product was chromatographed on silica gel, eluting withmethylene chloride, to yield pure product (152 mg, 72%) as a yellow oil.

C.

The nitrile produced in step B (1.2 g, 3.37 mmol) was dissolved inglacial acetic acid (30 ml). To this solution we added water (120 μl,6.67 mmol) followed by titanium tetrachloride (760 μl, 6.91 mmol), whichresulted in an exotherm. The solution was then refluxed for two hours,cooled and poured into 1N HCl. The aqueous layer was extracted withmethylene chloride. The organic layer was backwashed with 1N NaOH, driedover magnesium sulfate and filtered over a plug of silica gel. The plugwas first eluted with methylene chloride to remove unreacted startingmaterials, and then with ethyl acetate to yield compound 201. The ethylacetate was evaporated to yield pure compound 201 (1.0 g, 77%).

Example 8 Synthesis of p38 Inhibitor Compound 110

A.

The starting nitrile (3.76 g, 11.1 mmol) was first dissolved in glacialacetic acid (20 ml). To this solution we added titanium tetrachloride(22.2 mmol) and water (22.2 mmol) and heated the solution to reflux for1 hour. The reaction mixture was then cooled and diluted in water/ethylacetate. The organic layer was then isolated, washed with brine anddried over magnesium sulfate. The organic layer was then filtered andevaporated in vacuo. The resulting crude product was chromatographed onsilica gel eluting with 5% methanol in methylene chloride to afford pureproduct as a yellow foam (2.77 g, 70%)

B.

The amide produced in step A (1.54 g, 4.3 mmol) was dissolved in toluene(20 ml). We then added N,N-dimethylformamide dimethylacetal (1.53 g,12.9 mmol), heated the resulting solution for 10 minutes then allowed itto cool to room temperature. The reaction was then evaporated in vacuoand the residue was chromatographed on silica gel eluting with 2-5%methanol in methylene chloride. The recovered material was thendissolved in hot ethyl acetate. The solution was allowed to coolresulting in the crystallization of pure product as a yellow solid (600mg, 40%). Additional material (˜800 mg) was available from the motherliquor.

C.

The bromide from step B (369 mg, 1 mmol) was dissolved in THF (10 ml).We then added Diisobutylaluminum hydride (1.0M solution, 4 mmol),stirred the reaction at room temperature for 10 minutes, and thenquenched the reaction with methanol (1 ml). A saturated solution ofRochelle salts was then added and the mixture was extracted with ethylacetate. The organic layer was isolated, dried over magnesium sulfate,evaporated and the residue was chromatographed on silica gel elutingwith 1-3% methanol in methylene chloride to afford a bright orange solid(85 mg, 23% yield).

D.

The bromide produced in step C (35.2 mg, 0.1 mmol) was dissolved inxylene (12 ml). To this solution we added thiophenol (0.19 mmol)followed by tributyltin methoxide (0.19 mmol). The resulting solutionwas heated to reflux for 10 minutes, followed by the addition ofpalladium tetrakis(triphenylphosphine) (0.020 mmol). The reaction washeated and monitored for the disappearance of the bromide startingmaterial. The reaction was then cooled to room temperature and passedthrough a plug of silica gel. The plug was eluted initially withmethylene chloride to remove excess tin reagent and then with 5%methanol in ethyl acetate to elute the p38 inhibitor. The filtrate wasconcentrated and then re-chromatographed on silica gel using 5% methanolin ethyl acetate as eluant affording pure compound 110 (20 mg, 52%).

Example 9 Synthesis of p38 Inhibitor Compound 202

A.

The starting nitrile (2.32 g, 12 mmol) was dissolved in DMF (10 ml) atroom temperature. Sodium hydride (12 mmol) was then added resulting in abright yellow colored solution. After 15 minutes, a solution of 2,6dibromopyridine (2.36 gr., 10 mmol) in DMF (5 ml) was added, followed byPalladium tetrakis (triphenylphosphine) (1.0 mmol). The solution wasthen refluxed for 3 hours. The reaction was next cooled to roomtemperature and diluted with ethyl acetate. The organic layer wasisolated, washed with water and brine, dried over magnesium sulfate,filtered and evaporated in vacuo to a crude oil. Flash columnchromatography eluting with 10% ethyl acetate in hexane afforded product(1.45 g, 42%) as a white solid.

B.

The bromo compound produced in step A (1.77 g, 5.2 mmol) was dissolvedin toluene (20 ml) and the resulting solution was degassed. Under anitrogen atmosphere, a solution of phenylboronic acid (950 mg, 7.8 mmol)in ethanol (4 ml) and a solution of sodium carbonate (1.73 g, 14 mmol)in water (4 ml) were added. The reaction mixture was heated to refluxfor one hour and then was cooled to room temperature. The reaction wasdiluted with ethyl acetate and washed with water and brine. The organiclayer was then dried with magnesium sulfate, filtered and concentratedin vacuo. The residue was purified on silica gel eluting with 30% ethylacetate in hexane to afford product as a white solid (1.56 g, 88%).

C.

The nitrile from step B (700 mg, 2.07 mmol) was dissolved inconcentrated sulfuric acid (10 ml) and heated to 80° C. for 1 hour. Thereaction was then cooled to room temperature and the pH was adjusted to8 using 6N sodium hydroxide. The mixture was next extracted with ethylacetate. The organic layer was isolated, dried with magnesium sulfateand evaporated in vacuo to yield compound 202 as a yellow foam (618 mg,84%).

Example 10 Synthesis of Compound 410

A.

In a flame-dried 100 ml round-bottomed flask, 2.28 g (93.8 mmol) ofmagnesium chips were added to 50 ml of anhydrous tetrahydrofuran. Onecrystal of iodine was added forming a light brown color. To the solutionwas added 1.5 ml of a 10.0 ml (79.1 mmol) sample of2-bromo-5-fluorotoluene. The solution was heated to reflux. The browncolor faded and reflux was maintained when the external heat source wasremoved indicating Grignard formation. As the reflux subsided, another1.0-1.5 ml portion of the bromide was added resulting in a vigorousreflux. The process was repeated until all of the bromide had beenadded. The olive-green solution was externally heated to reflux for onehour to ensure complete reaction. The solution was cooled in an ice-bathand added via syringe to a solution of 9.3 ml (81.9 mmol) of trimethylborate in 100 ml of tetrahydrofuran at −78° C. After the Grignardreagent had been added, the flask was removed from the cooling bath andthe solution was stirred at room temperature overnight. Thegrayish-white slurry was poured into 300 ml of H₂O and the volatileswere evaporated in vacuo. HCl (400 ml of 2N solution) was added and themilky-white mixture was stirred for one hour at room temperature. Awhite solid precipitated. The mixture was extracted with diethyl etherand the organic extract was dried (MgSO₄) and evaporated in vacuo toafford 11.44 g (94%) of the boronic acid as a white solid.

B.

In a 100 ml round-bottomed flask, 7.92 g (33.4 mmol) of2,6-dibromopyridine was dissolved in 50 ml of anhydrous toluene forminga clear, colorless solution. 4-fluoro-2-methylbenzene boronic acid (5.09g, 33.1 mmol) produced in step A was added forming a white suspension.Thallium carbonate (17.45 g, 37.2 mmol) was added followed by acatalytic amount (150 mg) of Pd(PPh₃)₄. The mixture was heated to refluxovernight, cooled, and filtered over a pad of silica gel. The silica waswashed with CH₂Cl₂ and the filtrate was evaporated to afford a whitesolid. The solid was dissolved in a minimal amount of 50% CH₂Cl₂/hexaneand chromatographed on a short column of silica gel using 30%CH₂Cl₂/hexane to afford 6.55 g (74%) of the2-bromo-6-(4-fluoro-2-methylphenyl)pyridine as a white solid.

C.

In a 50 ml round-bottomed flask, 550 mg (2.07 mmol) of2-bromo-6-(4-fluoro-2-methylphenyl)pyridine produced in step B wasdissolved in 30 ml of anhydrous tetrahydrofuran forming a clear,colorless solution. 2,6-difluoroaniline (2.14 ml, 2.14 mmol) was addedfollowed by 112 mg (2.79 mmol) of a 60% NaH suspension in mineral oil.Gas evolution was observed along with a mild exotherm. The solution washeated to reflux overnight and then cooled. The reaction mixture waspoured in 10% NH₄Cl and extracted with CH₂Cl₂. The organic extract wasdried (MgSO₄) and evaporated in vacuo to afford a brown oil that was amixture of the product and starting material. The material waschromatographed on a short column of silica gel using 50% CH₂Cl₂/hexaneto afford 262 mg (40%) of2-(2,6-difluorophenyl)-6-(4-fluoro-2-methylphenyl)pyridine as acolorless oil.

D.

In a 100 ml round-bottomed flask, 262 mg (834 mmol) of2-(2,6-difluorophenyl)-6-(4-fluoro-2-methylphenyl)pyridine produced instep C was dissolved in 30 ml of anhydrous CHCl₃ forming a clear,colorless solution. Chlorosulfonyl isocyanate (1.0 ml, 11.5 mmol) wasadded and the light yellow solution was stirred at room temperatureovernight. Water (˜30 ml) was added causing a mild exotherm and vigorousgas evolution. After stirring overnight, the organic layer wasseparated, dried (MgSO₄) and evaporated in vacuo to afford a brown oilthat was a mixture of the product and starting material. The materialwas chromatographed on a short column of silica gel using 10%EtOAc/CH₂Cl₂. The recovered starting material was re-subjected to thereaction conditions and purified in the same manner to afford a total of205 mg (69%) of the urea as a white solid.

Example 11 Synthesis of Compound 138

Compound 103 (106 mg, 0.25 mmol) was dissolved in THF (0.5 ml) and tothis solution was added triethylamine (35 μl, 0.25 mmol) followed by andexcess of formaldehyde (37% aqueous solution, 45 mg). The reaction wasallowed to stir at room temperature overnight. The reaction mixture wasthen rotovapped under reduced pressure and the residue was dissolved inmethylene chloride and applied to a flash silica gel column. The columnwas eluted with 2% methanol in methylene chloride to yield pure product(78 mg, 70% yield).

Example 12 Synthesis of Prodrugs of Compound 103

A.

Compound 138 (1 equivalent) is dissolved in methylene chloride and tothis solution is added triethylamine (1 equivalent) followed bydibenzylphosphonyl chloride (1 equivalent). The solution is stirred atroom temperature and monitored by TLC for consumption of startingmaterial. The methylene chloride layer is then diluted with ethylacetate and washed with 1N HCl, saturated sodium bicarbonate andsaturated NaCl. The organic layer is then dried, rotovapped and thecrude product is purified on silica gel. The pure product is thendissolved in methanol and the dibenzyl esters are deprotected with 10%palladium on charcoal under a hydrogen atmosphere. When the reaction ismonitored as complete, the catalyst is filtered over celite and thefiltrate is rotovapped to yield the phosphate product.B.

Compound 103 (210 mg, 1.05 mmol) was dissolved in THF (2 ml) and cooledto −50° C. under a nitrogen atmosphere. To this solution was addedlithium hexamethyldisilazane (1.1 mmol) followed by chloroacetylchloride (1.13 mmol). The reaction was removed from the cooling bath andallowed to warm to room temperature, after which time the reaction wasdiluted with ethyl acetate and quenched with water. The organic layerwas washed with brine, dried and rotovapped to dryness. The crudeproduct was flash chromatographed on silica gel using 25% ethyl acetatein hexane as eluant to yield 172 mg (70%) of pure desired product, whichwas used as is in the next reactions.

C.

The chloroacetyl compound is dissolved in methylene chloride and treatedwith an excess of dimethyl amine. The reaction is monitored by TLC andwhen complete all volatiles are removed to yield desired product.

Example 13 Synthesis of Compounds 34 and 117

A.

The nitrile from Example 5, step A (300 mg, 1.0 mmol) was dissolved inethanol (10 ml) and to this solution was added thiourea (80.3 mg, 1.05mmol). The reaction was brought to reflux for 4 hours at which point TLCindicated that all starting material was consumed. The reaction wascooled and all volatiles were removed under reduced pressure, and theresidue was dissolved in acetone (10 ml).

To this solution was then added 2,5-difluoronitrobenzene (110 μl, 1.01mmol) followed by potassium carbonate (200 mg, 1.45 mmol) and water (400μl). The reaction was allowed to stir at room temperature overnight. Thereaction was then diluted with methylene chloride (25 ml) and filteredthrough a cotton plug. All volatiles were removed under reduced pressureand the residue was flashed chromatographed on silica gel eluting with agradient from 10%-25% ethyl acetate in hexane to yield the desiredproduct (142 mg, 33%).

B.

The nitrile product from Step A (142 mg, 0.33 mmol) was mixed withconcentrated sulfuric acid (2 ml), heated to reflux for 1 hour and thenallowed to cool to room temperature. The mixture was then diluted withethyl acetate and carefully neutralized with saturated potassiumcarbonate solution (aqueous). The layers were separated and the organiclayer was washed with water, brine and dried over magnesium sulfate. Themixture was filtered and evaporated to dryness. The residue was used inthe next step without further purification (127 mg, 85% yield).

C.

The amide from the step B (127 mg, 0.28 mmol) was dissolved in THF (3ml) and to this solution was added dimethylformamide dimethylacetal (110μl, 0.83 mmol). The reaction was heated to reflux for 5 minutes thencooled to room temperature. All volatiles were removed in vacuo and theresidue was flash chromatographed on silica gel eluting with 2.5%methanol in methylene chloride to yield pure desired compound 34 (118mg, 92%).

D.

A solution of nickel dichloride hexahydrate (103 mg, 0.44 mol) in amixture of benzene/methanol (0.84 mL/0.84 ml) was added to a solution ofcompound 34 (100.8 mg, 0.22 mmol) in benzene (3.4 ml) and this solutionwas cooled to 0° C. To this solution was then added sodium borohydride(49 mg, 1.3 mmol). The reaction was stirred while allowing to warm toroom temperature. The reaction was evaporated in vacuo and the residuewas flash chromatographed eluting with 2% methanol in methylene chlorideto yield pure desired product, compound 117 (21 mg, 25% yield).

Example 14 Synthesis of Compounds 53 and 142

A.

The product indicated in the above reaction was synthesized using theprocedure in example 1 step B using chloropyridazine (359 mg, 1.21 mmol)and 2,4 difluorothiophenol (176 mg, 1.21 mmol). The product was obtainedafter flash silica gel chromatography (451 mg, 92%).

B.

The above reaction was carried out as described in Example 1, step C,using 451 mg of starting material and 5 ml of concentrated sulfuric acidto yield the indicated product (425 mg, 90%).C.

The reaction above was carried out as described in Example 1, step D,using starting amide (410 mg, 0.96 mmol) and dimethylformamidedimethylacetal (3 mmol). The reaction was heated at 50° C. for 30minutes and worked up as described previously. Compound 53 was obtained(313 mg, 75%).

D.

Compound 34 (213, 0.49 mmol) was dissolved in THF (10 ml), cooled to 0°C. and to this solution was added Borane in THF (1M, 0.6 mmol). Thereaction was stirred for 30 minutes quenched with water and diluted withethyl acetate. The organic layer was washed with water and brine, driedand rotovapped. The residue was purified on silica gel eluting with agradient of 1% to 5% methanol in methylene chloride to afford compound142 (125 mg, 57%).

Example 15 Cloning of p38 Kinase in Insect Cells

Two splice variants of human p38 kinase, CSBP1 and CSBP2, have beenidentified. Specific oligonucleotide primers were used to amplify thecoding region of CSBP2 cDNA using a HeLa cell library (Stratagene) as atemplate. The polymerase chain reaction product was cloned into thepET-15b vector (Novagen). The baculovirus transfer vector,pVL-(His)6-p38 was constructed by subcloning a XbaI-BamHI fragment ofpET15b-(His)6-p38 into the complementary sites in plasmid pVL1392(Pharmingen).

The plasmid pVL-(His)6-p38 directed the synthesis of a recombinantprotein consisting of a 23-residue peptide (MGSSHHHHHHSSGLVPRGSHMLE,where LVPRGS represents a thrombin cleavage site) fused in frame to theN-terminus of p38, as confirmed by DNA sequencing and by N-terminalsequencing of the expressed protein. Monolayer culture of Spodopterafrugiperda (Sf9) insect cells (ATCC) was maintained in TNM-FH medium(Gibco BRL) supplemented with 10% fetal bovine serum in a T-flask at 27°C. Sf9 cells in log phase were co-transfected with linear viral DNA ofAutographa califonica nuclear polyhedrosis virus (Pharmingen) andtransfer vector pVL-(His)6-p38 using Lipofectin (Invitrogen). Theindividual recombinant baculovirus clones were purified by plaque assayusing 1% low melting agarose.

Example 16 Expression and Purification of Recombinant p38 Kinase

Trichoplusia ni (Tn-368) High-Five™ cells (Invitrogen) were grown insuspension in Excel-405 protein free medium (JRH Bioscience) in a shakerflask at 27° C. Cells at a density of 1.5×10⁶ cells/ml were infectedwith the recombinant baculovirus described above at a multiplicity ofinfection of 5. The expression level of recombinant p38 was monitored byimmunoblotting using a rabbit anti-p38 antibody (Santa CruzBiotechnology). The cell mass was harvested 72 hours after infectionwhen the expression level of p38 reached its maximum.

Frozen cell paste from cells expressing the (His)₆-tagged p38 was thawedin 5 volumes of Buffer A (50 mM NaH2PO4 pH 8.0, 200 mM NaCl, 2 mMβ-Mercaptoethanol, 10% Glycerol and 0.2 mM PMSF). After mechanicaldisruption of the cells in a microfluidizer, the lysate was centrifugedat 30,000×g for 30 minutes. The supernatant was incubated batchwise for3-5 hours at 4° C. with Talon™ (Clontech) metal affinity resin at aratio of 1 ml of resin per 2-4 mgs of expected p38. The resin wassettled by centrifugation at 500×g for 5 minutes and gently washedbatchwise with Buffer A. The resin was slurried and poured into a column(approx. 2.6×5.0 cm) and washed with Buffer A+5 mM imidazole.

The (His)₆-p38 was eluted with Buffer A+100 mM imidazole andsubsequently dialyzed overnight at 4° C. against 2 liters of Buffer B,(50 mM HEPES, pH 7.5, 25 mM β-glycerophosphate, 5% glycerol, 2 mM DTT).The His₆ tag was removed by addition of at 1.5 units thrombin(Calbiochem) per mg of p38 and incubation at 20° C. for 2-3 hours. Thethrombin was quenched by addition of 0.2 mM PMSF and then the entiresample was loaded onto a 2 ml benzamidine agarose (AmericanInternational Chemical) column.

The flow through fraction was directly loaded onto a 2.6×5.0 cmQ-Sepharose (Pharmacia) column previously equilibrated in Buffer B+0.2mM PMSF. The p38 was eluted with a 20 column volume linear gradient to0.6M NaCl in Buffer B. The eluted protein peak was pooled and dialyzedovernight at 4° C. vs. Buffer C (50 mM HEPES pH 7.5, 5% glycerol, 50 mMNaCl, 2 mM DTT, 0.2 mM PMSF).

The dialyzed protein was concentrated in a Centriprep (Amicon) to 3-4 mland applied to a 2.6×100 cm Sephacryl S-100HR (Pharmacia) column. Theprotein was eluted at a flow rate of 35 ml/hr. The main peak was pooled,adjusted to 20 mM DTT, concentrated to 10-80 mgs/ml and frozen inaliquots at −70° C. or used immediately.

Example 17 Activation of p38

P38 was activated by combining 0.5 mg/ml p38 with 0.005 mg/ml DD-doublemutant MKK6 in Buffer B+10 mM MgCl2, 2 mM ATP, 0.2 mM Na2VO4 for 30minutes at 20° C. The activation mixture was then loaded onto a 1.0×10cm MonoQ column (Pharmacia) and eluted with a linear 20 column volumegradient to 1.0 M NaCl in Buffer B. The activated p38 eluted after theADP and ATP. The activated p38 peak was pooled and dialyzed againstbuffer B+0.2 mM Na2VO4 to remove the NaCl. The dialyzed protein wasadjusted to 1.1M potassium phosphate by addition of a 4.0M stocksolution and loaded onto a 1.0×10 cm HIC (Rainin Hydropore) columnpreviously equilibrated in Buffer D (10% glycerol, 20 mMβ-glycerophosphate, 2.0 mM DTT)+1.1MK2HPO4. The protein was eluted witha 20 column volume linear gradient to Buffer D+50 mM K2HPO4. The doublephosphorylated p38 eluted as the main peak and was pooled for dialysisagainst Buffer B+0.2 mM Na2VO4. The activated p38 was stored at −70° C.

Example 18 P38 Inhibition Assays

A. Inhibition of Phosphorylation of EGF Receptor Peptide

This assay was carried out in the presence of 10 mM MgCl2, 25 mMβ-glycerophosphate, 10% glycerol and 100 mM HEPES buffer at pH 7.6. Fora typical IC50 determination, a stock solution was prepared containingall of the above components and activated p38 (5 nM). The stock solutionwas aliquotted into vials. A fixed volume of DMSO or inhibitor in DMSO(final concentration of DMSO in reaction was 5%) was introduced to eachvial, mixed and incubated for 15 minutes at room temperature. EGFreceptor peptide, KRELVEPLTPSGEAPNQALLR, a phosphoryl acceptor inp38-catalyzed kinase reaction (1), was added to each vial to a finalconcentration of 200 μM. The kinase reaction was initiated with ATP (100μM) and the vials were incubated at 30° C. After 30 minutes, thereactions were quenched with equal volume of 10% trifluoroacetic acid(TFA).

The phosphorylated peptide was quantified by HPLC analysis. Separationof phosphorylated peptide from the unphosphorylated peptide was achievedon a reverse phase column (Deltapak, 5 μm, C18 100D, part no. 011795)with a binary gradient of water and acteonitrile, each containing 0.1%TFA. IC50 (concentration of inhibitor yielding 50% inhibition) wasdetermined by plotting the % activity remaining against inhibitorconcentration.

B. Inhibition of ATPase Activity

This assay was carried out in the presence of 10 mM MgCl2, 25 mMβ-glycerophosphate, 10% glycerol and 100 mM HEPES buffer at pH 7.6. Fora typical Ki determination, the Km for ATP in the ATPase activity ofactivated p38 reaction was determined in the absence of inhibitor and inthe presence of two concentrations of inhibitor. A stock solution wasprepared containing all of the above components and activated p38 (60nM). The stock solution was aliquotted into vials. A fixed volume ofDMSO or inhibitor in DMSO (final concentration of DMSO in reaction was2.5%) was introduced to each vial, mixed and incubated for 15 minutes atroom temperature. The reaction was initiated by adding variousconcentrations of ATP and then incubated at 30° C. After 30 minutes, thereactions were quenched with 50 μl of EDTA (0.1 M, final concentration),pH 8.0. The product of p38 ATPase activity, ADP, was quantified by HPLCanalysis.

Separation of ADP from ATP was achieved on a reversed phase column(Supelcosil, LC-18, 3 μm, part no. 5-8985) using a binary solventgradient of following composition: Solvent A-0.1 M phosphate buffercontaining 8 mM tetrabutylammonium hydrogen sulfate (Sigma Chemical Co.,catalogue no. T-7158), Solvent B-Solvent A with 30% methanol.

Ki was determined from the rate data as a function of inhibitor and ATPconcentrations. The results for several of the inhibitors of thisinvention are depicted in Table 6 below:

TABLE 6 Compound K_(i) (μM) 1 >20 2 15 3 5.0 5 2.9 6 0.4

Other p38 inhibitors of this invention will also inhibit the ATPaseactivity of p38.

C. Inhibition of IL-1, TNF, IL-6 and IL-8 Production in LPS-StimulatedPBMCs

Inhibitors were serially diluted in DMSO from a 20 mM stock. At least 6serial dilutions were prepared. Then 4× inhibitor stocks were preparedby adding 4 μl of an inhibitor dilution to 1 ml of RPMI1640 medium/10%fetal bovine serum. The 4× inhibitor stocks contained inhibitor atconcentrations of 80 μM, 32 μM, 12.8 μM, 5.12 μM, 2.048 μM, 0.819 μM,0.328 μM, 0.131 μM, 0.052 μM, 0.021 μM etc. The 4× inhibitor stocks werepre-warmed at 37° C. until use.

Fresh human blood buffy cells were separated from other cells in aVacutainer CPT from Becton & Dickinson (containing 4 ml blood and enoughDPBS without Mg²⁺/Ca²⁺ to fill the tube) by centrifugation at 1500×g for15 min. Peripheral blood mononuclear cells (PBMCs), located on top ofthe gradient in the Vacutainer, were removed and washed twice withRPMI1640 medium/10% fetal bovine serum. PBMCs were collected bycentrifugation at 500×g for 10 min. The total cell number was determinedusing a Neubauer Cell Chamber and the cells were adjusted to aconcentration of 4.8×10⁶ cells/ml in cell culture medium (RPMI1640supplemented with 10% fetal bovine serum).

Alternatively, whole blood containing an anti-coagulant was useddirectly in the assay.

We placed 100 μl of cell suspension or whole blood in each well of a96-well cell culture plate. Then we added 50 μl of the 4× inhibitorstock to the cells. Finally, we added 50 μl of a lipopolysaccharide(LPS) working stock solution (16 ng/ml in cell culture medium) to give afinal concentration of 4 ng/ml LPS in the assay. The total assay volumeof the vehicle control was also adjusted to 200 μl by adding 50 μl cellculture medium. The PBMC cells or whole blood were then incubatedovernight (for 12-15 hours) at 37° C./5% CO2 in a humidified atmosphere.

The next day the cells were mixed on a shaker for 3-5 minutes beforecentrifugation at 500×g for 5 minutes. Cell culture supernatants wereharvested and analyzed by ELISA for levels of IL-1b (R & D Systems,Quantikine kits, #DBL50), TNF-∀ (BioSource, #KHC3012), IL-6 (Endogen,#EH2-IL6) and IL-8 (Endogen, #EH2-IL8) according to the instructions ofthe manufacturer. The ELISA data were used to generate dose-responsecurves from which IC50 values were derived.

Results for the kinase assay (“kinase”; subsection A, above), IL-1 andTNF in LPS-stimulated PBMCs (“cell”) and IL-1, TNF and IL-6 in wholeblood (“WB”) for various p38 inhibitors of this invention are shown inTable 7 below:

cell cell WB WB WB cmpd kinase IL-1 TNF IL-1 TNF IL-6 # IC50 IC50 IC50IC50 IC50 IC50 2 + N.D. N.D. N.D. N.D. N.D. 3 + N.D. N.D. N.D. N.D. N.D.5 + N.D. N.D. N.D. N.D. N.D. 6 ++ ++ + N.D. N.D. N.D. 7 + + + N.D. N.D.N.D. 8 + + + N.D. N.D. N.D. 9 + + + N.D. N.D. N.D. 10 + N.D. N.D. N.D.N.D. N.D. 11 + + + N.D. N.D. N.D. 12 ++ ++ ++ + + + 13 + + + N.D. N.D.N.D. 14 + ++ + N.D. N.D. N.D. 15 + ++ ++ N.D. N.D. N.D. 16 ++ + ++ N.D.N.D. N.D. 17 + + + N.D. N.D. N.D. 18 + + + N.D. N.D. N.D. 19 + + + N.D.N.D. N.D. 20 ++ + + N.D. N.D. N.D. 21 ++ ++ + N.D. N.D. N.D. 22 + + +N.D. N.D. N.D. 23 ++ ++ + + + + 24 ++ ++ ++ + + N.D. 25 ++ ++ + N.D.N.D. N.D. 26 + +++ ++ + + + 27 ++ + + + + + 28 ++ ++ ++ N.D. N.D. N.D.29 ++ ++ ++ N.D. N.D. N.D. 30 + + + + N.D. N.D. 31 + + + N.D. N.D. N.D.32 ++ + ++ + + + 33 ++ ++ ++ + + + 34 + + + N.D. N.D. N.D. 35 ++++ + + + + 36 + + + + + + 37 ++ ++ + + + + 38 +++ +++ ++ ++ ++ ++ 39++ + + N.D. N.D. N.D. 40 ++ ++ + N.D. N.D. N.D. 41 +++ +++ +++ N.D. N.D.N.D. 42 + N.D. N.D. N.D. N.D. N.D. 43 ++ + + N.D. N.D. N.D. 44 ++ + +N.D. N.D. N.D. 45 ++ N.D. N.D. N.D. N.D. N.D. 46 ++ + + N.D. N.D. N.D.47 ++ ++ + N.D. N.D. N.D. 48 ++ ++ + N.D. N.D. N.D. 49 ++ +++ + + + +50 + N.D. N.D. N.D. N.D. N.D. 51 ++ N.D. N.D. N.D. N.D. N.D. 52 ++ N.D.N.D. N.D. N.D. N.D. 53 +++ +++ +++ +++ +++ +++ 101 ++ +++ +++ + + ++ 102+++ +++ +++ + ++ ++ 103 +++ +++ +++ + ++ ++ 104 ++ ++ ++ + + + 105++ + + N.D. N.D. N.D. 106 +++ +++ +++ + ++ ++ 107 ++ + + N.D. N.D. N.D.109 +++ +++ +++ + + ++ 108 +++ ++ +++ ++ +++ +++ 110 ++ + + N.D. N.D.N.D. 111 ++ + + N.D. N.D. N.D. 112 ++ ++ + + + + 113 +++ +++ ++ + + +114 +++ +++ +++ ++ ++ +++ 115 +++ +++ +++ + + + 116 +++ +++ ++ + + + 117+++ +++ +++ ++ ++ +++ 118 ++ ++ ++ + + + 119 ++ N.D. N.D. N.D. N.D. N.D.120 N.D. ++ + + + + 121 +++ +++ ++ + + + 122 ++ ++ + + + + 123 ++ ++++ + + + 124 + + + N.D. N.D. N.D. 125 +++ +++ +++ + + + 126 + ++ + N.D.N.D. N.D. 127 +++ +++ +++ ++ ++ +++ 128 + + + N.D. N.D. N.D. 129 +++ ++++++ ++ + ++ 130 +++ ++ + N.D. N.D. N.D. 131 +++ +++ +++ N.D. N.D. N.D.132 +++ +++ ++ N.D. N.D. N.D. 133 +++ +++ +++ N.D. N.D. N.D. 134 +++++ + N.D. N.D. N.D. 135 +++ ++ + + + + 136 +++ +++ +++ + + ++ 137 ++++++ ++ + + ++ 138 ++ +++ ++ + + +++ 139 +++ +++ + + + + 140 +++ +++ +++++ + ++ 141 +++ +++ +++ + + + 142 +++ +++ +++ +++ +++ +++ 143 +++ +++++ + + + 144 +++ +++ ++ + + ++ 145 +++ +++ +++ +++ +++ +++ 201 ++ + + ++++ + 203 + N.D. N.D. N.D. N.D. N.D. 204 + N.D. N.D. N.D. N.D. N.D.205 + N.D. N.D. N.D. N.D. N.D. 206 ++ + + N.D. N.D. N.D. 207 + N.D. N.D.N.D. N.D. N.D. 208 N.D. ++ N.D. N.D. N.D. N.D. 209 N.D. + N.D. N.D. N.D.N.D. 202/301 +++ ++ ++ + + + 302 +++ +++ ++ + + + 303 + + + + + +304 + + + + + + 305 +++ +++ + + + + 306 ++ ++ + + + + 307 +++ ++ + + + +308 + N.D. N.D. N.D. N.D. N.D. 309 ++ ++ ++ + + + 310 ++ + + N.D. N.D.N.D. 311 ++ + + N.D. N.D. N.D. 312 +++ ++ + + + + 313 ++ + + N.D. N.D.N.D. 314 + N.D. N.D. N.D. N.D. N.D. 315 + N.D. N.D. N.D. N.D. N.D. 316 +N.D. N.D. N.D. N.D. N.D. 317 + + + N.D. N.D. N.D. 318 ++ N.D. N.D. N.D.N.D. N.D. 319 + N.D. N.D. N.D. N.D. N.D. 320 +++ ++ ++ N.D. N.D. N.D.321 + N.D. N.D. N.D. N.D. N.D. 322 ++ + + N.D. N.D. N.D. 323 ++ ++ ++N.D. N.D. N.D. 324 ++ ++ + N.D. N.D. N.D. 325 +++ +++ ++ + + + 326 +N.D. N.D. N.D. N.D. N.D. 327 ++ N.D. N.D. N.D. N.D. N.D. 328 + N.D. N.D.N.D. N.D. N.D. 329 ++ ++ + + + + 330 + N.D. N.D. N.D. N.D. N.D. 331 +N.D. N.D. N.D. N.D. N.D. 332 ++ ++ + + + + 333 ++ + + N.D. N.D. N.D.334 + N.D. N.D. N.D. N.D. N.D. 335 ++ + + + + + 336 + N.D. N.D. N.D.N.D. N.D. 337 + N.D. N.D. N.D. N.D. N.D. 338 + N.D. N.D. N.D. N.D. N.D.339 + N.D. N.D. N.D. N.D. N.D. 340 + N.D. N.D. N.D. N.D. N.D. 341 ++ ++++ N.D. N.D. N.D. 342 + N.D. N.D. N.D. N.D. N.D. 343 + N.D. N.D. N.D.N.D. N.D. 344 + N.D. N.D. N.D. N.D. N.D. 345 + N.D. N.D. N.D. N.D. N.D.346 ++ + + + + + 347 + N.D. N.D. N.D. N.D. N.D. 348 + N.D. N.D. N.D.N.D. N.D. 349 + ++ + + + + 350 + ++ + N.D. N.D. N.D. 351 + + + N.D. N.D.N.D. 352 + + N.D. N.D. N.D. N.D. 353 ++ + + N.D. N.D. N.D. 354 + N.D.N.D. N.D. N.D. N.D. 355 + N.D. N.D. N.D. N.D. N.D. 356 + N.D. N.D. N.D.N.D. N.D. 357 + N.D. N.D. N.D. N.D. N.D. 358 ++ + + N.D. N.D. N.D. 359 +N.D. N.D. N.D. N.D. N.D. 360 + N.D. N.D. N.D. N.D. N.D. 361 ++ ++ + N.D.N.D. N.D. 362 +++ ++ ++ + + + 363 +++ +++ ++ + + + 364 +++ +++ ++ + + +365 N.D. N.D. N.D. N.D. N.D. N.D. 366 + N.D. N.D. N.D. N.D. N.D. 367N.D. N.D. N.D. N.D. N.D. N.D. 368 N.D. N.D. N.D. N.D. N.D. N.D. 369 N.D.N.D. N.D. N.D. N.D. N.D. 370 N.D. N.D. N.D. N.D. N.D. N.D. 371 N.D. N.D.N.D. N.D. N.D. N.D. 372 N.D. N.D. N.D. N.D. N.D. N.D. 373 N.D. N.D. N.D.N.D. N.D. N.D. 374 ++ N.D. N.D. N.D. N.D. N.D. 375 +++ N.D. N.D. N.D.N.D. N.D. 376 +++ N.D. N.D. N.D. N.D. N.D. 377 +++ N.D. N.D. N.D. N.D.N.D. 378 +++ N.D. N.D. N.D. N.D. N.D. 379 +++ N.D. N.D. N.D. N.D. N.D.380 ++ N.D. N.D. N.D. N.D. N.D. 381 ++ N.D. N.D. N.D. N.D. N.D. 382 +++N.D. N.D. N.D. N.D. N.D. 383 +++ N.D. N.D. N.D. N.D. N.D. 384 ++ N.D.N.D. N.D. N.D. N.D. 385 ++ N.D. N.D. N.D. N.D. N.D. 386 + N.D. N.D. N.D.N.D. N.D. 387 + N.D. N.D. N.D. N.D. N.D. 388 +++ N.D. N.D. N.D. N.D.N.D. 389 ++ N.D. N.D. N.D. N.D. N.D. 390 + N.D. N.D. N.D. N.D. N.D. 391++ N.D. N.D. N.D. N.D. N.D. 392 ++ N.D. N.D. N.D. N.D. N.D. 393 ++ N.D.N.D. N.D. N.D. N.D. 394 +++ N.D. N.D. N.D. N.D. N.D. 395 +++ N.D. N.D.N.D. N.D. N.D. 396 +++ N.D. N.D. N.D. N.D. N.D. 397 + N.D. N.D. N.D.N.D. N.D. 398 N.D. N.D. N.D. N.D. N.D. N.D. 399 +++ N.D. N.D. N.D. N.D.N.D. 1301 +++ N.D. N.D. N.D. N.D. N.D. 401 +++ ++ ++ + + + 402 +++ ++++++ + + + 403 +++ +++ +++ + + ++ 404 +++ +++ +++ + + + 405 +++ +++ ++N.D. N.D. N.D. 406 ++ ++ + N.D. N.D. N.D. 407 ++ ++ + N.D. N.D. N.D. 408+++ +++ ++ N.D. N.D. N.D. 409 +++ +++ +++ + + ++ 410 +++ +++ +++ ++ ++++ 411 +++ +++ +++ + + + 412 N.D. N.D. N.D. N.D. N.D. N.D.

For kinase IC50 values, “+++” represents <0.1 μM, “++” representsbetween 0.1 and 1.0 μM, and “+” represents >1.0 μM. For cellular IL-1and TNF values, “+++” represents <0.1 μM, “++” represents between 0.1and 0.5 μM, and “+” represents >0.5 μM. For all whole blood (“WB”) assayvalues, “+++” represents <0.25 μM, “++” represents between 0.25 and 0.5μM, and “+” represents >0.5 μm. In all assays indicated in the tableabove, “N.D.” represents value not determined.

Other p38 inhibitors of this invention will also inhibit phosphorylationof EGF receptor peptide, and the production of IL-1, TNF and IL-6, aswell as IL-8 in LPS-stimulated PBMCs or in whole blood.

D. Inhibition of IL-6 and IL-8 Production in IL-1-Stimulated PBMCs

This assay was carried out on PBMCs exactly the same as above exceptthat 50 μl of an IL-1b working stock solution (2 ng/ml in cell culturemedium) was added to the assay instead of the (LPS) working stocksolution.

Cell culture supernatants were harvested as described above and analyzedby ELISA for levels of IL-6 (Endogen, #EH2-IL6) and IL-8 (Endogen,#EH2-IL8) according to the instructions of the manufacturer. The ELISAdata were used to generate dose-response curves from which IC50 valueswere derived.

Results for p38 inhibitor compound 6 are shown in Table 8 below:

TABLE 8 Cytokine assayed IC₅₀ (μM) IL-6 0.60 IL-8 0.85E. Inhibition of LPS-Induced Prostaglandin Endoperoxide Synthase-2(PGHS-2, or COX-2) Induction in PBMCs

Human peripheral mononuclear cells (PBMCs) were isolated from freshhuman blood buffy coats by centrifugation in a Vacutainer CPT (Becton &Dickinson). We seeded 15×10⁶ cells in a 6-well tissue culture dishcontaining RPMI 1640 supplemented with 10% fetal bovine serum, 50 U/mlpenicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine. Compound 6(above) was added at 0.2, 2.0 and 20 μM final concentrations in DMSO.Then we added LPS at a final concentration of 4 ng/ml to induce enzymeexpression. The final culture volume was 10 ml/well.

After overnight incubation at 37° C., 5% CO₂, the cells were harvestedby scraping and subsequent centrifugation, then the supernatant wasremoved, and the cells were washed twice in ice-cold DPBS (Dulbecco'sphosphate buffered saline, BioWhittaker). The cells were lysed on icefor 10 min in 50 μl cold lysis buffer (20 mM Tris-HCl, pH 7.2, 150 mMNaCl, 1% Triton-X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 2%aprotinin (Sigma), 10 μg/ml pepstatin, 10 μg/ml leupeptin, 2 mM PMSF, 1mM benzamidine, 1 mM DTT) containing 1 μl Benzonase (DNAse from Merck).The protein concentration of each sample was determined using the BCAassay (Pierce) and bovine serum albumin as a standard. Then the proteinconcentration of each sample was adjusted to 1 mg/ml with cold lysisbuffer. To 100 μl lysate an equal volume of 2×SDS PAGE loading bufferwas added and the sample was boiled for 5 min. Proteins (30 μg/lane)were size-fractionated on 4-20% SDS PAGE gradient gels (Novex) andsubsequently transferred onto nitrocellulose membrane by electrophoreticmeans for 2 hours at 100 mA in Towbin transfer buffer (25 mM Tris, 192mM glycine) containing 20% methanol. The membrane was pretreated for 1hour at room temperature with blocking buffer (5% non-fat dry milk inDPBS supplemented with 0.1% Tween-20) and washed 3 times in DPBS/0.1%Tween-20. The membrane was incubated overnight at 4° C. with a 1:250dilution of monoclonal anti-COX-2 antibody (Transduction Laboratories)in blocking buffer. After 3 washes in DPBS/0.1% Tween-20, the membranewas incubated with a 1:1000 dilution of horseradishperoxidase-conjugated sheep antiserum to mouse Ig (Amersham) in blockingbuffer for 1 h at room temperature. Then the membrane was washed again 3times in DPBS/0.1% Tween-20 and an ECL detection system (SuperSignal™CL-HRP Substrate System, Pierce) was used to determine the levels ofexpression of COX-2.

Results of the above mentioned assay indicate that compound 6 inhibitsLPS induced PGHS-2 expression in PBMCs.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the methods of this invention.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: each of Q₁ andQ₂ are independently selected from 5-6 membered aromatic carbocyclic orheterocyclic ring systems, or 8-10 membered bicyclic ring systemsconsisting of aromatic carbocyclic rings, aromatic heterocyclic rings ora combination of an aromatic carbocyclic ring and an aromaticheterocyclic ring; wherein: Q₁ is substituted with 1 to 4 substituents,independently selected from N(R′)C(O)R⁴; N(R′)C(O)OR⁴; N(R′)C(O)C(O)R⁴;N(R′)S(O₂)R⁴; N(R′)R⁴; N(R⁴)₂; OR⁴; OC(O)R⁴; OP(O)₃H₂; N═CH—N(R′)₂;3,4-methylenedioxy; —NH—C(O)—O—CH₂-4-pyridine; —NH—C(O)CH₂-morpholine;—NH—C(O)CH₂-piperazine; or —NH—C(O)CH₂-pyrrolidine; and Q₂ is optionallysubstituted with up to 4 substituents, independently selected from halo;C₁-C₃ straight or branched alkyl optionally substituted with NR′₂, OR′,CO₂R′, S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR′₂; O—(C₁-C₃)-alkyloptionally substituted with NR′₂, OR′, CO₂R′, S(O₂)N(R′)₂, N═CH—N(R′)₂,R³, or CONR′₂; NR′₂; OCF₃; CF₃; NO₂; CO₂R′; CON(R′)₂; R³; OR³; NHR³;SR³; C(O)R³; C(O)N(R′)R³; C(O)OR³; SR′; S(O₂)N(R′)₂; SCF₃; N═CH—N(R′)₂;CN; —NH—C(═NH)—NH₂; —CH₂—NH—C(═NH)—NH₂; or —CH₂—NH-imidazole; wherein R′is selected from hydrogen, (C₁-C₃)-alkyl; (C₂-C₃)-alkenyl or alkynyl;phenyl or phenyl substituted with 1 to 3 substituents independentlyselected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl orethyl; R³ is selected from a 5-6 membered aromatic carbocyclic orheterocyclic ring system; and R⁴ is (C₁-C₄)-alkyl optionally substitutedwith N(R′)₂, OR′, CO₂R′, CON(R′)₂, or SO₂N(R²)₂; or a 5-6 memberedcarbocyclic or heterocyclic ring system optionally substituted withN(R′)₂, OR′, CO₂R′, CON(R′)₂, or SO₂N(R²)₂; each R is independentlyselected from hydrogen, —R², —N(R²)₂, —OR², SR², —C(O)—N(R²)₂,—S(O_(2)—N(R) ²)₂, or —C(O)—OR², wherein two adjacent R are optionallybound to one another and, together with each Y to which they arerespectively bound, form a 4-8 membered carbocyclic or heterocyclicring; R² is selected from hydrogen, (C₁-C₃)-alkyl, or (C₂-C₃) -alkenyl;each optionally substituted with -N(R′)₂, —OR′, SR′, —C(O)—N(R′)₂,—S(O₂)—N(R′)₂, —C(O)—OR′, or R³; Y is C; and R₁ is selected fromhydrogen, (C₁-C₃)-alkyl, OH, or O—(C₁-C₃)-alkyl.
 2. The compoundaccording to claim 1, wherein Q₁ is selected from phenyl or pyridylcontaining 1 to 3 substituents independently selected from3,4-methylenedioxy, —N(CH₃)₂, —NH—S(O)₂-phenyl,—NH—C(O)O—CH₂-4-pyridine, —NH—C(O)CH₂-morpholine, —NH—C(O)CH₂—N(CH₃)₂,—NH—C(O)CH₂-piperazine, —NH—C(O)CH₂-pyrrolidine,—NH—C(O)C(O)-morpholine, —NH—C(O)C(O)-piperazine,—NH—C(O)C(O)-pyrrolidine, —O—C(O)CH₂—N(CH₃)₂, or —O—(CH₂)₂—N(CH₃)₂ andwherein at least one of said substituents is in the ortho position. 3.The compound according to claim 2, wherein Q₁ contains at least twosubstituents, both of which are in the ortho position.
 4. A compound ofthe formula:

or a pharmaceutically acceptable salt thereof, wherein: each of Q₁ andQ₂ are independently selected from 5-6 membered aromatic carbocyclic orheterocyclic ring systems, or 8-10 membered bicyclic ring systemsconsisting of aromatic carbocyclic rings, aromatic heterocyclic rings ora combination of an aromatic carbocyclic ring and an aromaticheterocyclic ring; wherein Q₁ is selected from:

Q₂ is optionally substituted with up to 4 substituents, independentlyselected from halo; C₁-C₃ straight or branched alkyl optionallysubstituted with NR′₂, OR′, CO₂R′, S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, orCONR′₂; O—(C₁-C₃)-alkyl optionally substituted with NR′₂, OR′, CO₂R′,S(O₂)N(R′)₂, N═CH—N(R′)₂, R³, or CONR′₂; NR′₂; OCF₃; CF₃; NO₂; CO₂R′;CON(R′)₂; R₃; OR³; NHR³; SR³; C(O)R³; C(O)N(R′)R³; C(O)OR³; SR′;S(O₂)N(R′)₂; SCF₃; N═CH—N(R′)₂; CN; —NH—C(═NH)—NH₂; —CH₂—NH—C(═NH)—NH₂;or —CH₂—NH-imidazole; wherein R′ is selected from hydrogen,(C₁-C₃)-alkyl; (C₂-C₃)-alkenyl or alkynyl; phenyl or phenyl substitutedwith 1 to 3 substituents independently selected from halo, methoxy,cyano, nitro, amino, hydroxy, methyl or ethyl; R³ is selected from a 5-6membered aromatic carbocyclic or heterocyclic ring system; and R⁴ is(C₁-C₄)-alkyl optionally substituted with N(R′)₂, OR′, CO₂R′, CON(R′)₂,or SO₂N(R²)₂; or a 5-6 membered carbocyclic or heterocyclic ring systemoptionally substituted with N(R′)₂, OR′, CO₂R′, CON(R′)₂, or SO₂N(R²)₂;each R is independently selected from hydrogen, —R₂, —N(R₂)₂, —OR², SR²,—C(O)—N(R²)₂, —S(O₂)—N(R²)₂, or —C(O)—OR², wherein two adjacent R areoptionally bound to one another and, together with each Y to which theyare respectively bound, form a 4-8 membered carbocyclic or heterocyclicring; R² is selected from hydrogen, (C₁-C₃)-alkyl, or (C₂-C₃) -alkenyl;each optionally substituted with —N(R′)₂, —OR′, SR′, —C(O)—N(R′)₂,—S(O₂)—N(R′)₂, —C(O)—OR′, or R³; Y is C; and R₁ is selected fromhydrogen, (C₁-C₃)-alkyl, OH, or O—(C₁-C₃)-alkyl.
 5. The compoundaccording to claim 4, wherein Q₁ is selected from 2-chloro-4,5methylenedioxy phenyl or 2-chloro-4-(N-2-morpholino-acetamido)phenyl. 6.The compound according to claim 1, wherein Q₂ is selected from phenyl orpyridyl and wherein Q₂ optionally contains up to 3 substituents, each ofwhich is independently selected from chloro, fluoro, bromo, methyl,ethyl, isopropyl, —OCH₃, —OH, —NH₂, —CF₃, —OCF₃, —SCH₃, —C(O)OH,—C(O)OCH₃, —CH₂NH₂, —N(CH₃)₂, —CH₂-pyrrolidine, —CH₂OH, —CH₂—N(CH₃)₂,—CH₂-piperazine, —NH—C(═NH)—NH₂, —CH₂—NH—C(═NH)—NH₂, and—CH₂—NH-imidazole.
 7. The compound according to claim 6, wherein, Q₂ isselected from:

unsubstituted 2-pyridyl or unsubstituted phenyl.
 8. The compoundaccording to claim 7, wherein Q₂ is selected from phenyl,2-isopropylphenyl, 3,4-dimethylphenyl, 2-ethylphenyl, 3-fluorophenyl,2-methylphenyl, 3-chloro-4-fluorophenyl, 3-chlorophenyl,2-carbomethoxylphenyl, 2-carboxyphenyl, 2-methyl-4-chlorophenyl,2-bromophenyl, 2-pyridyl, 2-methylenehydroxyphenyl, 4-fluorophenyl,2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2,4-difluorophenyl,2-hydroxy-4-fluorophenyl or 2-methylenehydroxy-4-fluorophenyl.
 9. Apharmaceutical composition comprising a compound according to claim 1 orclaim 4 and a pharmaceutically acceptable carrier.
 10. A method oftreating inflammatory diseases, destructive bone disorders,reperfusion/ischemia in stroke, myocardial ischemia, renal ischemia,cardiac hypertrophy, rheumatoid arthritis, inflammatory bowel disease,ulcerative colitis, or Crohn's disease in a patient, said methodcomprising administering to said patient a composition according toclaim
 9. 11. The method according to claim 10, wherein said method isused to treat an inflammatory disease selected from acute pancreatitis,chronic pancreatitis, asthma, allergies, or adult respiratory distresssyndrome.
 12. The method according to claim 10, wherein said method isused to treat rheumatoid arthritis, inflammatory bowel disease,ulcerative colitis, or Crohn's disease.
 13. The method according toclaim 10, wherein said method is used to treat a destructive bonedisorder selected from osteoarthritis, osteoporosis or multiplemyeloma-related bone disorder.
 14. The method according to claim 10,wherein said method is used to treat ischemia/reperfusion in stroke,myocardial ischemia, or renal ischemia.